Quinazoline Derivatives as Parp Inhibitors

ABSTRACT

The present invention provides compounds of formula (I), their use as PARP inhibitors as well as pharmaceutical compositions comprising said compnounds of formula (I) wherein R 1 , R 2 , R 3 , L, X, Y and Z have defined meanings.

FIELD OF THE INVENTION

The present invention relates to inhibitors of PARP and providescompounds and compositions containing the disclosed compounds. Moreover,the present invention provides methods of using the disclosed PARPinhibitors for instance as a medicine.

BACKGROUND OF THE INVENTION

The nuclear enzyme poly(ADP-riboso) polymerase-1 (PARP-1) is a member ofthe PARP enzyme family. This growing family of enzymes consist of PARPssuch as, for example: PARP-1, PARP-2, PARP-3 and Vault-PARP; andTankyrases (TANKs), such as, for example: TANK-1, TANK-2 and TANK-3.PARP is also referred to as poly(adenosine 5′-diphospho-ribose)polymerase or PARS (poly(ADP-ribose) synthetase).

PARP-1 is a major nuclear protein of 116 kDa consisting of threedomains: the N-terminal DNA binding domain containing two zinc fingers,the automodification domain and the C-terminal catalytic domain. It ispresent in almost-all eukaryotes. The enzyme synthesizespoly(ADP-ribose), a branched polymer that can consist of over 200ADP-ribose units. The protein acceptors of poly(ADP-ribose) are directlyor indirectly involved in maintaining DNA integrity. They includehistones, topoisomerases, DNA and RNA polymerases, DNA ligases, andCa²⁺- and Mg²⁺-dependent endonucleases. PARP protein is expressed at ahigh level in many tissues, most notably in the immune system, heart,brain and germ-line cells. Under normal physiological conditions, thereis minimal PARP activity. However, DNA damage causes an immediateactivation of PARP by up to 500-fold,

Tankyrases (TANKs) were identified as components of the human telomericcomplex. They have also been proposed to have a role in vesicletraffiecidng and may serve as scaffolds for proteins involved in variousother cellular processes. Telomeres, which are essential for chromosomemaintenance and stability, are maintained by teloinerase, a specializedreverse transcriptase. TANKs are (ADP-ribose)transferases with somefeatures of both signalling and cytoskeletal proteins. They contain thePAR? domain, which catalyses poly-ADP-ribosylation of substrateproteins, the sterile alpha motif, which is -shared with certainsignalling molecules and the ANK domain, which contains 24 ankyrinrepeats homologues to the cytoskeletal protein ankyrin. The ANK domaininteracts with a telomeric protein, Telomere Repeat binding Factor-1(TRP-1). These proteins were therefore named TRF1-interacting,ankyrin-related ADP-ribose polymerase (TANKs).

One of the more specific functions of TANK is the ADP-ribosylation ofTRF-1. Human telomere function requires two telomere-specific DNAbinding proteins, TRF-1 and TRF-2. TRF-2 protects chromosome ends, andTRF-1 regulates telomeric length. ADP-ribosylation inhibits the abilityof TRF-1 to bind to telomeric DNA. This poly-ADP-ribosylation of TRF-1releases TRF-1 from the telomeres, opening up the telomeric complex andallow access to telomerase. Therefore, TANK functions as a positiveregulator of telomere length., allowing-elongation of the telomeres bytelomerase.

Among the many functions attributed to PARP, and especially PARP-1, isits major role in facilitating DNA repair by ADP-ribosylation andtherefore coordinating a number of DNA repair proteins. As a result ofPART activation, NAD⁺ levels significantly decline. Extensive PARPactivation leads to severe depletion of NAD⁺ in cells suffering frommassive DNA damage. The short half-life of poly(ADP-ribose) results in arapid turnover rate. Once poly(ADP-ribose) is formed, it is quicldydegraded by the constitutively active poly(ADP-ribose) glycohydrolase(PARG), together with phosphodiesterase and (ADP-ribose) protein lyase.PARP and PARG form a cycle that converts a large amount off ADP toADP-ribose. In less than an hour, over-stimulation of PARP can cause adrop of NAD⁺ and ATP to less than 20% of the normal level. Such ascenario is especially detrimental during ischaemia when deprivation ofoxygen has already drastically compromised cellular energy output.Subsequent free radical production during reperfusion is assumed to be amajor cause of tissue damage. Part of the ATP drop, which is typical inmany organs during ischaemia and reperfusion, could be linked to NAD⁺depletion due to poly(ADP-ribose) turnover. Thus, PART or PARGinhibition is expected to preserve the cellular energy level therebypotentiating the survival of ischaemic tissues after insult

Poly(ADP-ribose) synthesis is also involved in the induced expression ofa number of genes essential.for inflammatory response. PARP inhibitorssuppress production of inducible nitric oxide syntbase (iNOS) inrnacrophages, P-type selectin and intercellular adhesion molecule-1(ICAM-1) in endothelial cells. Such activity underlies the stronganti-inlamnmation effects exhibited by PARP inhibitors. PARP inhibitionis able to reduce necrosis by preventing translocation and infiltrationof neutrophils to the injured tissues.

PARP is activated by damaged DNA fragments and, once activated,catalyzes the attachment of up to 100 ADP-ribose units to a variety ofnuclear proteins, including histones and PARP itself. During majorcellular stresses the extensive activation of PARP can rapidly lead tocell damage or death through depletion of energy stores. As fourmolecules of ATP are consumed for every molecule of NAD⁺regenerated,NAD⁺ is depleted by massive PARP activation, in the efforts tore-synthesize NAD⁺, ATP may also become depleted.

It has been reported that PAR-P activation plays a key role in bothNMDA- and NO-induced neurotoxicity. This has been demonstrated incortical cultures and in hippocampal slices wherein prevention oftoxicity is directly correlated to PARP ilnhibition potency. Thepotential role of PARP inhibitors in treating neurodegenerative diseasesand head trauma has thus been recognized even if the exact mechanism ofaction has not yet been elucidated.

Similarly, it has been demonstrated that single injections of PARPinhibitors have reduced the infarct size caused by iscbemia andreperfusion of the heart or skeletal muscle in rabbits. In thesestudies, a single injection of 3-amino-benzamide (10 mg/kg), either oneminute before occlusion or one minute before reprrfusion, caused similarreductions in infarct size in the heart (32-42%) while1,5-dihydroxyisoquinoline (1 mg/kg), another PARP inhibitor, reducedinfarct size by a comparable degree (38-48%. These results make itreasonable to assume that PARP inhibitors could salvage previouslyischaemic heart or reperfusion injury of skeletal muscle tissue.

PARP activation can also be used as a measure of damage followingneurotoxic insults resulting from exposure to any of the followinginducers like glutamate (via NMDA receptor stimulation), reactive oxygenintermediates, amyloid β-protein,N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its activemetabolite N-methyl-4-phenylpyridine (MPP⁺), which participate inpathological conditions such as stroke, Alzbeimer's disease andParkinsones disease. Other studies have continued to explore the role ofPARP activation in cerebellar granule cells in vitro and in MPTPneurotoxicity. Excessive neural exposure to glutamate, which serves asthe predominate central nervous system neurotransmitter and acts uponthe N-methyl D-aspartate NMDA) receptors and other subtype receptors,most often occurs as a result of stroke or other neurodegenerativeprocesses. Oxygen deprived neurons release glutamate in great quantitiesduring ischaemic brain insult such as during a stroke or heart attack.This excess release of glutamate in turn causes over-stimulation(excitotoxicity) of N-methyl-D-aspartate MA), AMPA, Kainate and MGRreceptors, which open ion channels and permit uncontrolled ion flow(e.g., Ca²⁺ and Na⁺ into the cells and K⁺ out of the cells) leading tooverstimulation of the neurons. The over-stimulated neurons secrete moreglutamate, creating a feedback loop or domino effect which ultimatelyresults in cell damage or death via the production of proteases, lipasesand free radicals. Excessive activation of glutamate receptors has beenimplicated in various neurological diseases and conditions includingepilepsy, stroke, Alzheimer's disease, Parkinson's disease, AmyotrophicLateral Sclerosis (ALS), Huntington's disease, schizophrenia, chronicpain, ischemia and neuronal loss following hypoxia, hypoglycemia,ischemia, trauma, and nervous insult Glutamate exposure and stimulationhas also been implicated as a basis for compulsive disorders,particularly drug dependence. Evidence includes findings in many animalspecies, as well as in cerebral cortical cultures treated with glutamateor NPDA, that glutamate receptor antagonists (i.e., compounds whichblock glutamate from binding to or activating its receptor) block neuraldamage following vascular stroke. Attempts to prevent excitotoxicity byblocking NMDA, AMPA, Kainate and MGR receptors have proven difficultbecause each receptor has multiple sites to which glutamate may bind andhence finding an effective mix of antagonists or universal antagonist toprevent binding of glutamate to all of the receptor and allow testing ofIisi theory, has been difficult Moreover, many of the compositions thatare effective in blocldng the receptors are alsoyoxic to animals. Assuch, there is presently no known effective treatment for glutamateabnormalities. The stimulation of NMDA receptors by glutamate, forexample, activates the enzyme neuronal nitric oxide synthase (nNOS),leading to the formation of nitric oxide (NO), which also mediatesneurotoxicity. NMDA neurotoxicity may be prevented by treatment withnitric oxide synthase (NOS) inhibitors or through targeted geneticdisruption of nNOS in vitro.

Another use for PARP inhibitors is the treatment of peripheral nerveinjuries, and the resultant pathological pain syndrome known asneuropathic pain, such as that induced by chronic constriction injury(CCI) of the common sciatic nerve and in which transsynaptic alterationof spinal cord dorsal horn characterized by hyperchromatosis ofcytoplasm and nucleoplasm (so-called “dark” neurons) occurs.

Evidence also exists that PARP inhibitors are useful for treatinginflammatory bowel disorders, such as colitis. Specifically, colitis wasinduced in rats by intraluminal administration of the haptentrinitrobenzene sulfonic acid in 50% ethanol. Treated rats received3-aminobenzanmide, a specific inhibitor of PARP activity. Inhibition ofPARP activity reduced the inflammatory response and restored themorphology and the energetic status of the distal colon.

Further evidence suggests that PARP inhibitors are useful for treatingarthritis. Further, PARP inhibitors appear to be useful for treatingdiabetes. PARP inhibitors have been shown to be useful for treatingendotoxic shock or septic shock.

PARP inhibitors have also been used to extend the lifespan andproliferative capacity of cells including treatment of diseases such asskin aging, Alzheimer's disease, atherosclerosis, osteoarrtis,osteoporosis, muscular dystrophy, degenerative diseases of skeletalmuscle involving replicative senescence, age-related musculardegeneration, immune senescence, AIDS, and other immune senescencedisease, and to alter gene expression of senescent cells.

It is also known that PARP inhibitors, such as 3-amino benzamide, affectoverall DNA repair in response, for example, to hydrogen peroxide orionizing radiation.

The pivotal role of PARP in the repair of DNA strand breaks is wellestablished, especially when caused directly by ionizing radiation or,indirectly after enzymatic repair of DNA lesions inauced by methylatingagents, topoisomerases I inhibitors and other chemotherapeutic agents ascisplatin and bleomycin. A variety of studies using “knockout” nice,trans-dominant inhibition models (over-expression of the DNA-bindingdomain), antisense and small molecular weight inhibitors havedemonstrated the role of PAPRP in repair and cell survival afterinduction of DNA damage. The inhibition of PARP enzymatic activityshould lead to an enhanced sensitivity of the tumor cells towards DNAdamaging treatments.

PARP inhibitors have been reported to be effective in radiosensitizing(hypoxic) tumor cells and effective in preventing tumor cells fromrecovering from potentially lethal and sublethal damage of DNA afterradiation therapy, presumably by their ability to prevent DNA strandbreak rejoining and by affecting several DNA damage signaling pathways.

PARP inhibitors have been used to treat cancer. In addition, U.S. Pat.No. 5,177,075 discusses several isoquinolines used for enhancing thelethal effects of ionizing radiation or chemotherapeutic agents on tumorcells. Weltin et al., “effect of 6(5-Phenanthridinone), an Inhibitor ofPoly(ADP-ribose) Polymerase, on Cultured Tumor Cells”, Oncol. Res.,6:9,399-403 (1994), discusses the inhibition of PARP activity, reducedproliferation of tumor cells, and a marked synergistic effect when tumorcells are co-treated with an alkylating drug.

Reviews of te state of the art has been published by Li and Zhang inIDrugs 2001, 4(7): 804-812, by Ame et al in Bioassays 2004, 26: 882-883and by Nguewa et al., in Progress in Biophysic & Molecular Biology 2005,88: 143-172.

There continues to be a need for effective and potent PARP inhibitors,and more particularly PARP-1 inhibitors which produce minimal sideeffects. The present invention provides compounds, compositions for, andmethods of, inhibiting PARP activity for treating cancer and/orpreventing cellular, tissue andlor organ damage resulting from celldamage or death due to, for example, necrosis or apoptosis. Thecompounds and compositions of the present invention are especiallyuseful in enhancing the effectiveness of chemotherapy and radiotherapywhere a primary effect of the treatment is that of causing DNA damage inthe targeted cells.

BACKGROUND PRIOR ART

GB 1062357 published on Mar. 22, 1967 discloses quinazolone derivativeshaving antihypertensive effects.

DE 2258561 published on Jun. 20, 1973 discloses substituted pyridinonederivatives with antihypertensive action.

EP 13612, published on Nov. 11, 1983, discloses substitutedpipexidinylalkylquinazoline derivatives. The described compounds areserotonin-antagonists.

EP 669919, published on Jun. 9, 1994, discloses dimethylbenzofurans anddimethylbenzopyrans as 5-HT₃ antagonists. More in particular compoundsNo. 8, 4, 5, 10, 11, 12, 13, 15, 16, 17 and 14 of the presentapplication are disclosed. U.S. Pat. No. 5,374,637, published on Dec.20, 1994, discloses benzamide derivatives. The disclosed compounds havegastrointestinal motility stimulating propres. In particular compoundsNo. 8, 6 and 9 of the present application are disclosed. EP 885190,published on Dec. 23, 1998 discloses 1,4-disubstituted piperidinederivatives having gastrokinetic properties. In particular compound No.7 of the present application is disclosed

EP 1036073, published on Jun. 17, 1999, discloses substitutedquinazolinedione derivatives. The described compounds have fundicrelaxation properties.

HP 1355888 published on 20 Jun. 2002 discloses quinazolinone derivativesas PARP inhibitors.

DESCRIPTION OF THE INVENTION

This invention concerns compounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

the dotted lines represent optional bonds;

X is >N— or >CH—;

—N═Y— is —N—C(O)— or —N═CR⁴—, wherein R⁴is hydroxy;

L is a direct bond or a bivalent radical selected from —C(O)—,—C(O)—NH—, —NH—, —C(O)—C₁₋₆alkanediyl-, —C(O)—O—C₁₋₆alkanediyl- or—C₁₋₆alkanediyl-;

R¹ is hydrogen, halo, C₁₋₆alkyloxy or C₁₋₆alkyl;

R² is hydrogen, hydroxy, C₁₋₆alkyloxy or aminocarbonyl;

when X is substituted with R² than R²taken together with -L-Z can form abivalent radical of formula

—C(O)—NH—CH₂—NR¹⁰—  (a-1)

wherein R¹⁰ is phenyl;

R³ is hydrogen, or C₁₋₆alkyloxy;

Z is amino, cyano or a radical selected from

wherein each R⁵, R⁶, R⁷ and R⁸ is independently selected from hydrogen,halo, amino, C₁₋₆alkyl or C₁₋₆alkyloxy; or

R⁷ and R⁸ taken together may form a bivalent radical of formula

—CH₂—CR⁹ ₂—O—  (c-1),

—(CH₂)₃—O—  (c-2),

—O—(CH₂)₂—O—  (c-3) or

—CH═CH—CH═CH—  (c-4)

wherein each R⁹ is independently selected from hydrogen or C₁₋₆alkyl;with the proviso thatwhen X is >N—, then Z is other than the radical (b-2) andwhen X is >CH— and L is —C(O)—NH— or

—C(O)—O—C₁₋₆alkanediyl- and Z is the radical (b-2) and R⁷ and R⁸ takentogether form a bivalent radical of formula (c-1), (c-2) or (c-3) thenR⁵ is other than chloro.

The compounds of formula (I) may also exist in their tautomeric forms.Such forms although not explicitly indicated in the above formula amintended to be included within the scope of the present invention.

A number of terms used in the foregoing definitions and hereinafter areexplained hereunder. These terms are sometimes used as such or incomposite terms.

As used in the foregoing definitions and hereinafter, halo is generic tofluoro, chloro, bromo and iodo; C₁₋₆alkyl defines straight and branchedchain saturated hydrocarbon radicals having from 1 to 6 carbon atomssuch as, e.g. methyl, ethyl, propyl, butyl, pentyl, hexyl,1-methylethyl, 2-methyipropyl, 2-methyl-butyl, 2-methylpentyl and thelike; C₁₋₆alkanediyl defines bivalent straight and branched chainedsaturated hydrocarbon radicals having from 1 to 6 carbon atoms such as,for example, methylene, 1,2-ethanediyl, 1,3-propanediyl 1,4-butanediyl,1,5-pentanediyl, 1,6-hexanediyl and the branched isomers thereof suchas, 2-methylpentanediyl, 3-methylpentanediyl, 2,2-dimethylbutanediyl,2,3-dimethylbutanediyl and the like.

The term “pharmaceutically acceptable salts” means pharmaceuticallyacceptable acid or base addition salts. The pharmaceutically acceptableacid or base addition salts as mentioned hereinabove are meant tocomprise the therapeutically active non-toxic acid and non-toxic baseaddition salt forms which the compounds of formula (I) are able to form.The compounds of formula (I) which have basic properties can beconverted in their pharmaceutically acceptable acid addition salts bytreating said base form with an appropriate acid. Appropriate acidscomprise, for example, inorganic acids such as bydrohalic acids, e.g.hydrochloric or hydrobroniic acid; sulfuric; nitric; phosphoric and thelike acids; or organic acids such as, for example, acetic, propanoic,hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e.butanedioic acid), maleic, fumaric, malic, tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.

The compounds of formula (I) which have acidic properties may beconverted in their pharmaceutically acceptable base addition salts bytreating said acid form with a suitable organic or inorganic base.Appropriate base salt forms comprise, for example, the ammonium salts,the alkali and earth alkaline metal salts, e.g. the lithium, sodium,potassium, magnesium, calcium salts and the like, salts with organicbases, e.g. the benzathine, N-methyl-D-glucamine, hydrabanine salts, andsalts with amino acids such as, for example, arginine, lysine and thelike.

The terms acid or base addition salt also comprise the hydrates and thesolvent addition forms which the compounds of formula (1) are able toform Examples of such forms ame e.g. hydrates, alcoholates and the like.

The term stereochemically isomenc forms of compounds of formula (I), asused hereinbefore, defines all possible compounds made up of the sameatoms bonded by the same sequence of bonds but having differentthree-dimensional structures which are not interchangeable, which thecompounds of formula (I) may possess. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms which saidcompound may possess Said mixture may contain all diastereomers and/orenantiomers of the basic molecular structure of said compound. ADstereochemically isomeric forms of the compounds of formula (I) both inpure form or in admixture with each other are intended to be embracedwithin the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprisethose compounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide, particularly those N-oxides whereinone or more of the piperidine- or piperazine nitrogens are N-oxidized.

Whenever used hereinafter, the term “compounds of formula (I)” is meantto include also the N-oxide forms, the phamaceutically acceptable acidor base addition salts and all stereoisomeric forms.

GB 1062357 discloses quinazolone derivatives having antihypertensiveeffects. DE 2258561 discloses substituted pyridinone derivatives withantihypertensive action. EP 13612 discloses substitutedpiperidinylalkylquinazoline derivatives that are serotonin-antagonists.EP 669919 discloses dimethylbenzofurans and dimethylbenzopyrans as 5-HT₃antagonists. U.S. Pat. No. 5,374,637 discloses benzamide derivativesthat have gastrointestinal motility stimulating properties. EP 885190discloses 1,4-disubstituted piperidine derivatives having gastrokineticproperties. EP 1036073 discloses substituted quinazolinedionederivatives that have fundic relaxation properties.

Unexpectedly, it has been found that the compounds of the presentinvention show PARP inhibitory activity.

A first group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

a) each X is >N—;

b) L is a bivalent radical selected from —C(O)—, —C(O)—NH—, —NH—,—C(O)—C₁₋₆alkanediyl-, —C(O)—O-C₁₋₆alkanediyl- or —C₁₋₆alkanediyl-;

c) R¹ is hydrogen;

d) R²is hydroxy, C₁₋₆alkyloxy or aminocarbonyl;

e) Z is amino, cyano or a radical selected from (b-1), (b-3), (b-4),(b-5), (b-6), (b-7), (b-8) or (b-9);

f) each R⁵ and R⁶ is independently selected from hydrogen or amino.

A second group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

a) X is >CH—;

b) L is a direct bond or a bivalent radical selected from —C(O)—, —NH—,—C(O)—C₁₋₆alkanediyl-, or —C₁₋₆alkanlediyl-;

c) Z is amino, cyano or a radical selected from (b-1), (b-3), (b-4),(b-5), (b-6), (b-7), (b-8) or (b-9);

d) each R⁵ is independently selected from hydrogen, fluoro, iodo, bromo,amino, C₁₋₆alkyl or C₁₋₆alkyloxy;

e) each R⁶ is independently selected from hydrogen, chloro, iodo, bromo,amino,

A third group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

a) L is a direct bond or a bivalent radical selected from —C(O)—, or—C(O)—NH—;

b) R² is hydrogen, hydroxy, or C₁₋₆aikyloxy;

c) Z is a radical selected from (b-2), (b-3), (b-4), (b-5), (b-6),(b-7), (b-8) or (b-9);

d) each R⁵, R⁶, R⁷ and R⁹ is independently selected from hydrogen, halo,C₁₋₆alkyl or C₁₋₆alkyloxy; or

e) R⁷ and R⁸ taken together may form a bivalent radical of formula(c-1), or (c-4).

A fourth group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

a) L is a direct bond or a bivalent radical selected from —C(O)—, or—C(O)—NH—, or —C(O)—O—C₁₋₆alkanediyl-;

b) R² is hydrogen, hydroxy, or C₁₋₆alkyloxy;

c) Z is a radical selected from (b-2), (b-3), (b-4), (b-5), (b-6),(b-7), (b-8) or (b-9);

d) each R⁵, R⁶, R⁷ and R⁹ is independently selected from hydrogen, halo,C₁₋₆alkyl or C₁₋₆alkyloxy; or

e) R⁷ and R⁸ taken together may form a bivalent radical of formula(c-1), (c-2), (c-3) or (c-4).

A fifth group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

a) L is a direct bond;

b) R¹ is hydrogen, halo or C₁₋₆alkyl;

c) R² is hydrogen;

d) R³ is hydrogen;

e) Z is a radical selected from (b-5) or (b-7);

f) each R⁵ is independently selected from hydrogen or halo.

A group of preferred compounds consists of those compounds of formula(I) wherein L is a direct bond or a bivalent radical selected from—C(O)—, —C(O)—NH—, or —C(O)—O—C₁₋₆alkanediyl-; R² is hydrogen, hydroxy,or C₁₋₆-alkyloxy; Z is a radical selected from (b-2), (b-3), (b-4),(b-5), (b-6), (b-7), (b-8) or (b-9); each R⁵, R⁶, R⁷ and R⁸ isindependently selected from hydrogen, halo, amrino, C₁₋₆alkyl orC₁₋₆alkyloxy; or R⁷ and R⁸ taken together may form a bivalent radical offormula (c-1), (c-2), (c-3) or (c4).

A group of more preferred compounds consists of those compounds offormula (I) wherein

L is a direct bond, R¹ is hydrogen, halo or C₁₋₆alkyl;

R² is hydrogen; R³ is hydrogen; Z is a radical selected from (b-5) or(b-7); and each R⁵ is independently selected from hydrogen or halo.

The most preferred compounds are compounds No. 35, No. 36, No. 39, No. 1and No. 43.

The compounds of formula (I) can be prepared according to the generalmethods described in EP 1036073, BP 885190, U.S. Pat. No. 5,374,637, EP669919 and EP13612. The starting materials and some of the intermediatesare known compounds and are commercially available or may be preparedaccording to conventional reaction procedures generally known in theart.

Some preparation methods will be described hereinafter in more detail.Other methods for obtaining final compounds of formula (I) are describedin the examples.

The compounds of formula (I), can be prepared by reacting anintermediate of formula (II), with an intermediate of formula (III),wherein W is an appropriate leaving group such as, for example, halo,e.g. fluoro, chloro, bromo or iodo, or a sulfonyloxy radical such asmethylsulfonyloxy,

4-methylphenylsulfonyloxy and the like. The reaction can be performed ina reaction-inert solvent such as, for example, an alcohol, e.g.methanol, ethanol, 2-methoxy-ethanol, propanol, butanol and the like; anether, e.g. 4, 4-dioxane, 1,1′-oxybispropane and the like; or a ketone,e.g. 4-methyl-2-pentanone, N,N-dimethylformamide, nitrobenzene and thelike. The addition of an appropriate base such as, for example, analkali or earth alkaline metal carbonate or hydrogen carbonate, e.g.triethylamine or sodium carbonate, may be utilized to pick up the acidwhich is liberated during the course of the reaction. A small amount ofan appropriate metal iodide, e.g., sodium or potassium iodide may beadded to promote the reaction. Stirring may enhance the rate of thereaction. The reaction may conveniently be carried out at a temperatureranging between room temperature and the reflux temperature of thereaction mixture and, if desired, the reaction may be carried out at anincreased pressure.

The compounds of formula (I) may also be converted into each other viaart-known reactions or functional group transformations. Some of suchtransformations are already described hereinabove. Other examples arehydrolysis of carboxylic esters to the corresponding carboxylic acid oralcohol; hydrolysis of amides to the corresponding carboxylic acids oramines; hydrolysis of nitrites to the corresponding amides; amino groupson irnidazole or phenyl may be replaced by a hydrogen by art-knowndiazotation reactions and subsequent replacement of the diazo-group byhydrogen; alcohols may be converted intoe.sters and ethers; primaryamines may be converted into secondary or tertiary amines; double bondsmay be hydrogenated to the corresponding single bond; an iodo radical ona phenyl group may be converted in to an ester group by carbon monoxideinsertion in the presence of a suitable palladium catalyst.

The present invention also relates to a compound of formula (I) asdefined above for use as a medicine.

The compounds of the present invention have PARP inhibiting propertiesas can be seen from the experimental part hereinunder.

The term “PARP” is used herein to mean a protein havingpoly-ADP-ribosylation activity. Within the meaning of this term, PARPencompass all proteins encoded by a parp gene, mutants thereof, andalternative slice proteins thereof. Additionally, as used herein, theterm “PARP” includes PARP analogues, homologues and analogues of otheranimals.

The term “PARP”, includes but is not limited to PARP-1. Within themeaning of this term PARP-2, PARP-3, Vault-PARP (PARP-4), PARP-7(TiPARP), PARP-8, PARP-9 (Bal), PARP-10, PARP-11, PARP-12, PARP-13,PARP-14, PARP-15, PARP-16, TANK-1, TANK-2, and TANK-3 may beencompassed.

Compounds that inhibit both PARP-1 and- tankyrase 2 can haveadvantageous properties in that they have enhanced growth inhibitingactivities in cancer cells.

The present invention also contemplates the use of compounds in thepreparation of a medicament for the treatment of any of the diseases anddisorders in an animal described herein, wherein said compounds arecompounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

the dotted lines represent optional bonds;

X is >N— or >CH—;

—N═Y— is —N—C(O)— or —N═CR⁴—, wherein R⁴is hydroxy;

L is a direct bond or a bivalent radical selected from —C(O)—,—C(O)—NH—, —NH—, —C(O)—C₁₋₆alkanediyl-, —C(O)—O—C₁₋₆alkanediyl- or—C₁₋₆alkanediyl-;

R¹ is hydrogen, halo, C₁₋₆alkyloxy or C₁₋₆alkyl;

R² is hydrogen, hydroxy, C₁₋₆alkyloxy or aminocarbonyl;

when X is substituted with R² than R²taken together with -L-Z can form abivalent radical of formula

—C(O)—NH—CH₂—NR¹⁰—  (a-1)

wherein R¹⁰ is phenyl;

R³ is hydrogen, or C₁₋₆alkyloxy;

Z is amino, cyano or a radical selected from

wherein each R⁵, R⁶, R⁷ and R⁸ is independently selected from hydrogen,halo, amino, C₁₋₆alkyl or C₁₋₆alkyloxy; or

R⁷ and R⁸ taken together may form a bivalent radical of formula

—CH₂—CR⁹ ₂—O—  (c-1),

—(CH₂)₃—O—  (c-2),

—O—(CH₂)₂—O—  (c-3) or

—CH═CH—CH═CH—  (c-4)

wherein each R⁹ is independently selected from hydrogen or C₁₋₆alkyl.

Furthermore, the invention also concerns the use of a compound asdescribed above for the manufacture of a medicament for the treatment ofa disorder mediated through PARP.

In particular, the invention concerns the use of a compound as describedabove for the manufacture of a medicament for the treatment of adisorder mediated through PARP.

Compounds that inhibit both PARP-1 and TANK-2 can have advantagesproperties in that they have enhanced growth inhibiting activities incancer cells.

In view of their PARP binding properties the compounds of the presentinvention may be used as reference compounds or tracer compounds inwhich case one of the atoms of the molecule may be replaced with, forinstance, a radioactive isotope.

To prepare the pharmaceutical compositions of this invention, aneffective amount of a particular compound, in base or acid addition saltform, as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, preferably, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of ine usualpharmaceutical media may be employed, such as, for example, tater,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets. Because of their ease in administration, tabletsand capsules represent the most advantageous oral dosage unit form, inwhich case solid pharmaceutical carriers are obviously employed. Forparenteral compositions, the carrier will usually comprise sterilewater, at least in large part, though other ingredients, to aidsolubility for example, may be include& Injectable solutions, forexample, may be prepared in which the carrier comprises saline solution,glucose solution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed In thecompositions suitable for percutaneous administration, the carrieroptionally comprises a penetration enhancing agent and/or a suitablewetting agent, optionally combined with suitable additives of any naturein minor proportions, which additives do not cause a significantdeleterious effect to the skin. Said additives may facilitate theadministration to the slin andlor may be helpful for preparing thedesired compositions. These compositions may be administered in variousways, e.g., as a transdermal patch, as a spot-on, as an ointment. It isespecially advantageous to formulate the aforementioned pharmaceuticalcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used in the specification andclaims herein refers to physically discrete units suitable as unitarydosages, each unit containing a predetermined quantity of activeingredient calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Examples of suchdosage unit forms are tablets (including scored or coated tablets),capsules, pills, powder packets, wafers, injectable solutions orsuspensions, teaspoonfuls, tablespoonfuls and the like, and segregatedmultiples thereof.

The compounds of the present invention can treat or prevent tissuedamage resulting from cell damage or death due to necrosis or apoptosis;can ameliorate neural or cardiovascular tissue damage, including thatfollowing focal ischernia, myocardial infarction, and reperfusioninjury; can treat various diseases and conditions caused or exacerbatedby PARP activity; can extend or increase the lifespan or proliferativecapacity of cells; can alter the gene expression of senescent cells; canradiosensitize and/or chemosensitize cells. Generally, inhibition ofPARP activity spares the cells from energy loss, preventing, in the caseof neural cells, irreversible depolarization of the neurons, and thus,provides neuroprotection.

For the foregoing reasons, the present invention further relates to amethod of administering a therapeutically effective amount of theabove-identified compounds in an amount sufficient to inhibit PARPactivity, to treat or prevent tissue damage resulting from cell damageor death due to necrosis or apoptosis, to effect a neuronal activity notmediated by NODA toxicity, to effect a neuronal activity mediated byNMDA toxicity, to treat neural tissue damage resulting from ischemia andreperfusion injury, neurological disorders and neurodegenerativediseases; to prevent or treat vascular stroke; to treat or preventcardiovascular disorders; to treat other conditions and/or disorderssuch as age- related muscular degeneration, AIIDS and other immunesenescence diseases, inflammation, gout, arthritis, atherosclerosis,cachexia, cancer, degenerative diseases of skeletal muscle involvingreplicative senescence, diabetes, head trauma, inflammatory boweldisorders (such as colitis and Crohn's disease), muscular dystrophy,osteoarthritis, osteoporosis, chronic and/or acute pain (such asneuropathic pain), renal failure, retinal ischemia, septic shock (suchas endotoxic shock), and slin aging, to extend the lifespan andproliferative capacity of cells; to alter gene expression of senescentcells; chemosensitize and/or radiosensitize (hypoxic) tumor cells. Thepresent invention also relates to treating diseases and conditions in ananimal which comprises administering to said animal a therapeuticallyeffective amount of the above-identified compounds.

In particular, the present invention relates to a method of treating,preventing or inhibiting a neurological disorder in an animal, whichcomprises administering to said animal a therapeutically effectiveamount of the above-identified compounds. The neurological disorder isselected from the group consisting of peripheral neuropathy caused byphysical injury or disease state, traumatic brain injury, physicaldamage to the spinal cord, stroke associated with brain damage, focalischernia, global ischemia, reperfusion injury, demyelinating diseaseand neurological disorder relating to neurodegeneration.

The present invention also contemplates the use of compounds of formula(I) for inhibiting PARP activity, for treating, preventing or inhibitingtissue damage resulting from cell damage or death due to necrosis orapoptosis, for treating, preventing or inhibiting a neurologicaldisorder in an animal.

The term “preventing neurodegeneration” includes the ability to preventneurodegeneration in patients newly diagnosed as having aneurodegenerative disease, or at risk of developing a new degenerativedisease and for preventing further neurodegeneration in patients who arealready suffering from or have symptoms of a neurodegenerative disease.

The term “treatment” as used herein covers any treatment of a diseaseand/or condition in an animal, particularly a human, and includes: (i)preventing a disease and/or condition from occurring in a subject whichmay be predisposed to the disease and/or condition but has not yet beendiagnosed as having it; (ii) inhibiting the disease and/or condition,ie., arresting its development; (iii) relieving the disease andlorcondition, i.e., causing regression of the disease and/or condition.

The term “radiosensitizer”, as used herein, is defined as a molecule,preferably a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of thecells to ionizing radiation and/or to promote the treatment of diseaseswhich are treatable with ionizing radiation. Diseases which aretreatable with ionizing radiation include neoplastic diseases, benignand malignant tumors, and cancerous cells. Ionizing radiation treatmentof other diseases not listed herein are also contemplated by the presentinvention.

The term “chemosensitize”, as used herein, is defined as a molecule,preferably a low molecular weight molecule, admnistered to animals intherapeutically effective amounts to increase the sensitivity of cellsto chemotherapy and/or promote the treatment of diseases which aretreatable with chemotherapeutics. Diseases which are treatable withchemotherapy include neoplastic diseases, benign and malignant tmors andcancerous cells. Chemotherapy treatment of other diseases not listedherein are also contemplated by the present invention.

The compounds, compositions and methods of the present invention areparticularly useful for treating or preventing tissue damage resultingfrom cell death or damage due to necrosis or apoptosis.

The compounds of the present invention can be “anti-cancer agents”,which term also encompasses “anti-tumor cell growth agents” and“anti-neoplastic agents”. For example, the methods of the invention areuseful for treating cancers and chemosensitizing and/or radiosensitizingtumor cells in cancers such as ACTTI-producing tumors, acute lymphocyticleukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex,bladder cancer, brain cancer, breast cancer, cervical cancer, chroniclymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer,cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer,Ewing's sarcoma gallbladder cancer, hairy cell leukemia, head &neckcancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, livercancer, lung cancer (small and/or non-small cell), malignant peritonealeff-sion, malignant pleural effusion, melanoma, mesothelioma, multiplemyeloma, neuroblastoma, non- Hodjkin's lymphoma, osteosarcoma, ovariancancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer,penile cancer, retinoblastoma, skin cancer, soft tissue sarcoma,squamous cell carcinomas, stomach cancer, testicular cancer, thyroidcancer, trophoblastic neoplasms, uterine cancer, vaginal cancer, cancerof the vulva and Wilm's tumor.

Hence the compounds of the present invention can be used as“radiosensitizer” and/or “chemosensitizer”.

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of ionizing radiation. Several mechanisms forthe mode of action of radiosensitizers have been suggested in theliterature including: hypoxic cell radiosensitizers ( e.g.,2-nitroimidazole compounds, and benzotriazine dioxide compounds)mimicking oxygen or alternatively behave like bioreductive agents underhypoxia; non-hypoxic cell radiosensitizers (e.g., halogenatedpyrrimidines) can be analogs of DNA bases and preferentially incorporateinto the DNA of cancer cells and thereby promote the radiation-inducedbreaking of DNA molecules and/or prevent the normal DNA repairmechanisms; and various other potential mechanisms of action have beenhypothesized for radiosensitizers in the treatment of disease. Manycancer treatment protocols currently employ radiosensitizers inconjunction with radiation of x-rays. Examples of xray activatedradiosensitizers include, but are not imrited to, the following:metronidazole, misonidazole, desmethyhisonidazole, pimonidazole,etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145,nicotinamide, 5-bromodeoxyuridine BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuxidine (FudR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives, tinetioporphyrin, pheoborbide-a, bacteriochlorophyll-a, naphthaloayanines,phthalocyanines, zinc phthalocyanine, and therapeutically effectiveanalogs and derivatives of the same.

Radiosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds which promote the incorporationof radiosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents which act on the tumor with or withoutadditional radiation; or other therapeutically effective compounds fortreating cancer or other disease. Rex Examples of additional therapeuticagents that may be used in conjunction with radiosensitizers include,but are not limited to: 5-fluorouracil, leucovorin, 5′-aminodeoxythymidine, oxygen, carbogen, red cell transfusions,perrfuorocarbons (egg., Fluosol 10 DA), 2,3-DPG, BW12C, calcium channelblockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, andLBSO. Examples of chemotherapeutic agents that may be used inconjunction with radiosensitizers include, but are not limited to:adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,docetaxel, doxorubicin, interferon (alpha, beta, gamna), interleukin 2,irinotecan, paclitaxel, topotecan, and therapeutically effective analogsand derivatives of the same.

Chemosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds which promote the incorporationof chemosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemothearpeutic agents which act on the thmor or other therapeuticallyeffective compounds for treating cancer or other disease. Examples ofadditional therapeutical agents that may be used in conjunction withchemosensitizers include, but are not limited to: methylating agents,toposisomerase I inhibitors and other chemotherapeutic agents such ascisplatin and bleomycin.

The compounds of formula (I) can also be used to detect or identify thePARP, and more in particular the PARP-1 receptor For that purpose thecompounds of formula (I) can be labeled. Said label can be selected fromthe group consisting of a radioisotope, spin label, antigen label,enzyme label fluorescent group or a chemiluminiscent group.

Those skilled in the art could easily determine the effective amountfrom the test results presented hereinafter. In general it iscontemplated that an effective amount would be from 0.001 mg/kg to 100mg/kg body weight, and in particular from 0.005 mg/kg to 10 mg/kg bodyweight. It may be appropriate to administer the required dose as two,three, four or more sub-doses at appropriate intervals throughout theday. Said sub-doses may be formulated as unit dosage forms, for example,containing 0.05 to 500 mg, and in particular 0.1 mg to 200 mg of activeingredient per unit dosage for m

Experimental Part

Hereinafter, “DCM” is defined as dichioromethane, “DMF” is defined asN,N-dimethylfonnannde, “MeOH” is defined as methanol, “MIK” is definedas methyl isobutyl keton, “MEK” is defined as methyl ethyl keton, “TEA”is defined as triethylamine and “THF” is defined as tetrahydrofuran.

A. Preparation of the Intermediate Compounds

EXAMPLE A1

a) Preparation of Intermediate 1

A mixture of 3-(1-piperazinyl)-1H-indazole (0.11 mol),chloro-acetonitrile (0.16 mol) and TEA (13 g) in toluene (200 ml) andacetonitile (200 ml) was stirred and refluxed for 3 hours. The cooledreaction mixture was washed with water (250 ml). The organic layer wasseparated, dried (MgSO₄), filtered and the solvent was evaporated. Theresidue was dissolved in trichloromethane and purified over silica on aglass filter (eluent: trichloromethane /MeOH 90/10). The purest fractionwas collected and the solvent was evaporated. The residue wascrystallized from acetonitrile. The crystals were filtered off anddried, yielding 26 g (99%) of intermediate 1, melting point 136° C.

b) Preparation of Intermediate 2

A mixture of intermediate 1 (0.11 mol) in NH₃/MeOH (600 ml) washydrogenated at 50° C. with Raney Nickel (4 g) as a catalyst. Afteruptake of H₂ (2 eq), the catalyst was filtered off and the filtrate wasevaporated. The residue was crystallized from acetontrile. The crystalswere filtered off and dried, yielding 21 g (77.5%) of intermediate 2,melting point 121° C.

EXAMPLE A2

a) Preparation of Intermediate 3

Phosphoryl chloride (110.9 ml) was added dropwise at 5° C. to DMF (81.5ml). The mixture was stirred until complete dissolution.4[(1-oxobutyl)amino]-benzoic acid, ethyl ester (0.34 mol) was added. Themnixture was stired at 100° C. for 15 hours, then cooled to roomtemperature and poured out into ice water. The precipitate was filteredoff, washed with water and dried, yielding 42.35 g (47%) of intermediate3.

b) Preparation of Intermediate 4

A mixture of intermediate 3 (0.1606 mol) in sodium methylate, 30%solution in MeOH (152.8 ml) and MeOH (400 ml) was stirred and refluxedfor 15 hours, then cooled and poured out into ice water. The precipitatewas filtered off, washed with water and taken up in DCM. The organiclayer was separated, dried (MgSO₄), filtered and the solvent wasevaporated till dryness, yielding 31.64 g (85%) of intermediate 4.

c) Preparation of Intermediate 5

Lithium tetrahydroaluminate (0.1288 mol) was added portionwise at 0° C.under N₂ flow to a solution of intermediate 4 (0.1288 mol) in BT (263ml). The mixture was stired for 30 min, poured out into ice water andextracted with DCM. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated till dryness, yielding 27.4 g(98%) of intermediate 5.

d) Preparation of Intermediate 6

Methanesulfonyl chloride (0.104 mol) was added dropwise at 0° C. underN₂ flow to a mixture of intermediate 5 (0.069 mol) and TEA (0.207 mol)in DCM (120 ml). The mixture was stirred at 0° C. for 4 hours. Thesolvent was evaporated till dryness (without heating). The product wasused without further purification, yielding 20.4 g of intermediate 6.

EXAMPLE A3

a) Preparation of Intermediate 7

4(2-aminoethyl)-1-piperazinecarboxylic acid, ethyl ester (0.0586 mol)and 2-(methylthio)-4(1H)-quinazolinone (0.0588 mol) were heated at 180°C. for 2 hours while stirring upon treatment with javelle water and thentaken up in DCM and MeOH. The solvent was evaporated till dryness. Theresidue was purified by column chromatography over silica gel (15-35 μm)(eluent: DCM/MeOH/NH₄OH 94/6/0.5). The pure fractions were collected andthe solvent was evaporated. The oily residue was crystallized fromdiethyl ether. The precipitate, was filtered off and dried, yieldingintermediate 7, melting point: 138° C.

b) Preparation of Intermediate 8

A mixture of intermediate 7 (0.0223 mol) and potassium hydroxide (0.223mol) in 2-propanol (100 ml) was stirred and refluxed for 4 days. Thesolvent was evaporated till dryness. The residue was taken up in MeORwhile stirng at 60° C. The salts were filtered off. The solvent wasevaporated, yielding 6.5 g of intermediate 8.

B. Preparation of the Final Compounds EXAMPLE B1 Preparation of Compound1

Intermediate 2 (0.000815 mol) and 6-chloro-2-(methylthio)-4(1H)-quinazolinone (0.00097 mol) were heated at 160° C. for 1 hour,then taken up in water and potassium carbonate 10% and extracted withDCM/MeOH 90/10. The organic layer was separated, dried (MgSO₄), filteredand the solvent was evaporated. The residue (0.3 g) was purified bycolumn chromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH92/8/0.5). The pure fractions were collected and the solvent wasevaporated. The residue was crystallized from MEK and DIPE. Theprecipitate was filtered off and dried, yielding 0.2 g (58%) of compound1, melting point 186° C.

EXAMPLE B2 Preparation of Compound 2

A mixture of 1-(3-aminopropyl)-4-(4-chlorophenyl)-4-piperidinol (0.015mol) and 2-chloro-4(1H)-quinazolinone (0.018 mol) in dimethylacetamiide(5 ml) was stored at 120° C. for 1 hour. The reaction mixture wascooled, dissolved in DCM and washed with aqueous ammonia The organiclayer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The residue was purified by column chromatography oversilica gel (eluent: DCM/(MeOH/NH₃) 92/8). The pure fractions werecollected and the solvent was evaporated. The residue was suspended inDIPE. The precipitate was filtered off and dried (vacuum; 70° C.),yielding 3.72 g (60%) of compound 2, melting point 178.4° C.

EXAMPLE B3 Preparation of Compound 3

A mixture of intermediate 6(0.0124 mol), intermediates (0.0137 mol) andpotassium carbonate (0.0373 mol) in DMF (80 ml) was stirred at 60° C.for 1 hour, poured out into ice water and stirred at room temperaturefor 30 min. The precipitate was filtered off, washed with water andtaken up in 2-propanone. The precipitate was filtered off and dried,yielding 1.5 g (26%) of compound 3, melting point 118° C.

TABLE F-1 lists the compounds that were prepared according to one of theabove Examples.

Co. No. 1; Ex. [B1]; mp. 186° C.

Co. No. 2; Ex. [B2]; mp. 178.4° C.

.H₂O (1:1); Co. No. 3; Ex. [B3]; mp. 118° C.

Co. No. 19; Ex. [B1]; mp. 206.3° C.

Co. No. 20; Ex. [B1]; mp. 164.6° C.

Co. No. 21; Ex. [B1]; mp. 193° C.

Co. No. 22; Ex. [B1]; mp. 254° C.

Co. No. 23; Ex. [B1]; mp. 243.1° C.

Co. No. 24; Ex. ]B1]; mp. 133.6° C.

Co. No. 25; Ex. [B1]; mp. 274.4° C.

.H₂O (2:1)•C₄H₄O₄ (2:1); Co. No. 26; Ex. [B1]; mp. 147.1° C.

Co. No. 27; Ex. [B1]; mp. 274° C.

Co. No. 28; Ex. [B1]; mp. 119.5° C.

Co. No. 29; Ex. [B1]; mp. 255.5° C.

Co. No. 30; Ex. [B1]; mp. 230° C.

.C₂H₂O₄ (1:2)•H₂O (1:1); Co. No. 31; Ex. [B1]; mp. 142° C.

.H₂O (1:1); Co. No. 32; Ex. [B1]; mp. 154° C.

.H₂O (1:5); Co. No. 33; Ex. [B1]; mp. 190° C.

.C₂H₂O₄ (1:2)•H₂O (1:2)Co. No. 34; Ex. [B1]; mp. 156° C.

Co. No. 35; Ex. [B1]; mp. >260° C.

.C₂H₂O₄ (1:2)•H₂O (1:1); Co. No. 36; Ex. [B1]; mp. 134° C.

.C₂H₂O₄ (1:1); Co. No. 37; Ex. [B1]; mp. 148° C.

.H₂O (1:1); Co. No. 38; Ex. [B1]; mp. 205° C.

Co. No. 39; Ex. [B1]; mp. 172° C.

Co. No. 40; Ex. [B1]

Co. No. 41; Ex. [B1]; mp. 242.2° C.

Co. No. 42; Ex. [B1]

Co. No. 43; Ex. [B1]; mp. 254° C.

Co. No. 44; Ex. [B1]; mp. 172° C.

.H₂O (2:1); Co. No. 45; Ex. [B2]; mp. 192.6° C.

.HCl (1:2); Co. No. 46; Ex. [B1]; mp. 253.8° C.

(B-CIS); Co. No. 18; Ex. [B2]; mp. 145.8° C.

Co. No. 4; EP 669919

Co. No. 5; EP669919

(CIS); Co. No. 6; US 5374637

Co. No. 7; EP 885190

Co. No. 8; EP 669919, US 5374637

Co. No. 9, US 5374637

Co. No. 10, EP 669919

Co. No. 11, EP 669919

Co. No. 12, EP 669919

Co. No. 13, EP 669919

Co. No. 14, EP 669919

Co. No. 15, EP 669919

Co. No. 16, EP 669919

Co. No. 17, EP 669919

Pharmacological Example In vitro Scintillation Proximity Assay (SPA) forPARP-1 Inhibitory Activity

Compounds of the present invention were tested in an in vitro assaybased on SPA technology (proprietary to Amersham Phannacia Biotech). Inprinciple, the assay relies upon the well established SPA technology forthe detection of poly(ADP-ribosyl)ation of biotinylated target proteins,i.e histones. This ribosylation is induced using nicked DNA activatedPARP-1 enzyme and [³H]-nicotinamide adenine dinucleotide ([I³H]-NAD⁺) asADP-ribosyl donor.

As inducer of PARP-1 enzyme activity, nicked DNA was prepared. For this,25 mg of DNA (supplier: Sigma) was dissolved in 25 ml DNAse buffer (10MM Tris-HCl, pH 7.4; 0.5 mg/ml Bovine Serum Albumine (MSA); 5 mMMgCl₂.6H₂O and 1 mM KCl) to which 50 μl DNAse solution (1 mg/ml 0.15 MNaCl) was added. After an incubation of 90 min. at 37° C., the reactionwas terminated by adding 1.45 g NaCl, followed by a further incubationat 58 ° C for 15 min. The reaction mixture was cooled on ice anddialysed at 4° C. for respectively 1.5 and 2 hours against 1.5 l of 0.2M KCl, and twice against 1.51 of 0.01 M KCl for 1.5 and 2 hrespectively. The mixture was aliquoted and stored at −20 ° C. Histones(1 mg/ml, type II-A, supplier: Sigma) were biotinylated using thebiotinylation kit of Amnersham and stored aliquoted at −20 ° C. A stocksolution of 100 mg/ml SPA poly(vinyl toluene) (PVT) beads (supplier:Amersham) was made in PBS. A stock solution of [³H]-NAD⁺ was made byadding 120 μl of [³H]-NAD⁺ (0.1 mCi/ml, supplier: NW to 6 ml incubationbuffer (50 mM Tris/HCl, pH 8; 0.2 nM DTT; 4 mM MgCl₂). A solution of 4MM NAD⁺ (supplier: Roche) was made in incubation buffer (from a 100 mMstock solution in water stored at −20° C.) The PARP-1 enzyme wasproduced using aft known techniques, i.e. cloning and expression of theprotein starting from human liver cDNA. Information concerning the usedprotein sequence of the PARP-1 enzyme including literature referencescan be found in the Swiss-Prot database under primary accession numberP09874. Biotinylated histones and PVT-SPA beads were mixed andpreincubated for 30 min. at room temperature. PARP-1 enzyme(concentration was lot dependent) was mixed with the nicked DNA and themixture was pre-incubated for 30 min. at 4° C. Equal parts of thishistones/PVT-SPA beads solution and PARP-1 enzyme/DNA solution weremixed and 75 μl of this mixture together with 1 μl of compound in DMSOand 25 μl of [³H]-NAD⁺ was added per well into a 96-wellmicrotiterplate. The final concentrations in the incubation mixture were2 μg/ml for the biotinylated histones, 2 mg/ml for the PVT-SPA beads, 2μg/ml for the nicked DNA and between 5-10 μg/ml for the PARP-1 enzyme.After incubation of the mixture for 15 min. at room temperature, thereaction was terminated by adding 100 μl of 4 mM NADE in incubationbuffer (final concentration 2 mM) and plates were mixed.

The beads were allowed to sediment for at least 15 min. and platestransferred to a TopCountNXT™ (Packard) for scintillation counting,values were expressed as counts per minute (cpm). For each experiment,controls (containing PARP-1 enzyme and DMSO without compound), a blankincubation (containing DMSO but no PARP-1 enzyme or compound) andsamples (containing PARP-1 enzyme and compound dissolved in DMSO) wererun in parallel. All compounds tested were dissolved and eventuallyfurther diluted in DMSO. In first instance, compounds were tested at aconcentration of 10⁻⁵M. When the compounds showed activity at 10⁻⁵M, adose-response curve was made wherein the compounds were tested atconcentrations between 10⁻⁵M and 10⁻⁸M. In each test, the blank valuewas subtracted from both the control and the sample values. The controlsample represented maximal PARP-1 enzyme activity. For each sample, theamount of cpm was expressed as a percentage of the mean cpm value of thecontrols. When appropriate, IC₅₀-values (concentration of the drug,needed to reduce the PARP-1 enzyme activity to 50% of the control) werecomputed using linear interpolation between the experimental points justabove and below the 50% level. Herein the effects of test compounds areexpressed as pIC₅₀ (the negative log value of the IC₅₀-value). As areference compound, 4-amino-1,8-naphthalimide was included to validatethe SPA assay. The tested compounds showed inhibitory activity at theinitial test concentration of 10⁻⁵ M (see Tabel-2).

In vitro Filtration Assay for PARP-1 Inhibitory Activity

Compounds of the present invention were tested in an in vitro filtrationassay assessing PARP-1 activity (triggered in the presence of nickedDNA) by means of its histone poly (ADP-ribosyl)ation activity using[³²P]-NAD as ADP-ribosyl donor. The radioactive ribosylated histoneswere precipitated by trchloroacetic acid (TCA) in 96-well filterplatesand the incorporated [³²P] measured using a scintillation counter

A mixture of histones (stock solution: 5 mg/ml in H₂O), NAD⁺ (stocksolution: 100 mM in H₂O), and [³²P]-NAD⁺ in incubation buffer (50 mMTris/HCl, pH 8; 0.2 mM DTT; 4 mM MgCl₂) was made. A mixture of thePARP-1 enzyme (5 -10 μg/ml) and nicked DNA was also made. The nicked DNAwas prepared as described in the in vitro SPA for PARP-1 inhibitoryactivity. Seventy-five μl of the PARP-1 enzyme/DNA mixture together with1 μl of compound in DMSO and 25 μl of histones-NAD⁺/[³²P]-NAD⁺ mixturewas added per well of a 96-well filterplate (0.45 μm, supplierMillpore). The final concentrations in the incubation mixture were 2μg/ml for the histones, 0.1 mM for the NAD⁺, 200 μM (0.5 μC) for the[³²P]-NAD⁺ and 2 μg/ml for the nicked DNA. Plates were incubated for 15min. at room temperature and the reaction was terminated by the additionof 10 μl ice cold 100% TCA followed by the addition of 10 μl ice-coldBSA solution (1% in H₂O). The protein fraction was allowed toprecipitate for 10 min. at 4° C. and plates were vacuum filtered. Theplates were subsequently washed with, for each well, 1 ml of 10% icecold TCA, 1 ml of 5% ice cold TCA and 1 ml of 5% TCA at roomtemperature. Finally 100 μl of scintillation solution (Microscint 40,Packard) was added to each well and the plates were transferred to aTopCountNXT™ (supplier: Packard) for scintillation counting and valueswere expressed as counts per minute (cpm). For each experiment, controls(containing PARP-1 enzyme and DMSO without compound), a blank incubation(containing DMSO but no PARP-1 enzyme or compound) and samples(containing PARP-1 enzyme and compound dissolved in DMSO) were run inparallel. All compounds tested were dissolved and eventually furtherdiluted in DMSO. In first instance, compounds were tested at aconcentration of 10⁻⁵M. When the compounds showed activity at 10⁻⁵M, adose-response curve was made wherein the compounds were tested atconcentrations between 10⁻⁵M and 10⁻⁸M. In each test, the blank valuewas subtracted from both the control and the sample values. The controlsample represented maximal PARP-1 enzyme activity. For each sample, theamount of cpm was expressed as a percentage of the mean cpm value of thecontrols. When appropriate, IC₅₀-values (concentration of the drug,needed to reduce the PARP-1 enzyme activity to 50% of the control) werecomputed using linear interpolation between the experimental points justabove and below the 50% level. Herein the effects of test compounds areexpressed as pIC₅₀ (the negative log value of the IC₅₀-value). As areference compound, 4-amino-1,8-naphthalimide was included to validatethe filtration assay. The tested compounds showed inhibitory activity atthe initial test concentration of 10⁻⁵M (see Tabel-2).

In vitro Scintillation Proximity Assay (SPA) for TANK-2 InhibitoryActivity

Compounds of the present invention were tested in an in vitro assaybased on SPA technology with Ni Flash plates (96 or 384 well).

In principle, the assay relies upon SPA technology for the detection ofauto-poly(ADP-ribosyl)ation of TANK-2 protein using [³H]-nicotinamideadenine dinucleotide ([³H]-NAD⁺) as ADP-ribosyl donor.

A stock solution of [³H]-NAD⁺/NAD was made by adding 64.6 μl of[³H⇄-NAD⁺ (0.1 mCi/ml, supplier Perkin Elmer) and 46.7 μl NAD-stock(10.7 mM, stored at −20° C., supplier Roche) to 1888.7 μl assay buffer(60 mM Tris/HCl, pH 7.4; 0.9 mM DTT; 6 mM MgCl₂). The TANK-2 enzyme wasproduced as described in EP1238063. 60 μL of assay buffer, together with1 μl of compound in DMSO, 20 μl of [³H]-NAD^(+/)NAD and 20 μl of TANK-2enzyme (final concentration 6 μg/ml) was added per well into a 96-wellNi-coated flash plate (Perlin Elmer). After incubation of the mixturefor 120 min. at room temperature, the reaction was terminated by adding60 μl of stopsolution (42.6 mg NAD in 6 ml H₂O). The plates were coveredwith a plate sealer and placed in a TopCountNXT™ (Packard) forscintillation counting. Values were expressed as counts per minute(cpm). For each experiment, controls (containing TANK-2 enzyme and DMSOwithout compound), a blank incubation (containing DMSO but no TANK-2enzyme or compound) and samples (containing TANK-2 enzyme and compounddissolved in DMSO) Were run in parallel. All compounds tested weredissolved and eventually further diluted in DMSO. In first instance,compounds were tested at a concentration of 10⁻⁵M. When the compoundsshowed activity at 10⁻⁵M, a dose-response curve was made wherein thecompounds were tested at concentrations between 10⁻⁵M and 10⁻⁸CM. Ineach test, the blank value was subtracted from both the control and thesample values. The control sample represented maximal TANK-2 enzymeactivity For each sample, the amount of cpm was expressed as apercentage of the mean cpr value of the controls. When appropriate,IC₅₀-values (concentration of the drug, needed to reduce the TANK-2enzyme activity to 50% of the control) were computed using linearinterpolation between the experimental points just above and below the50% level. Herein the effects of test compounds are expressed as pIC₅₀(the negative log value of the IC₅₀-value). As reference compounds,3-aminobenzamide and 4-amino-1,8-naphtalimide were included to validatethe SPA assay. Herein the assay was described using 96-well plates. Inthe assay using 384-well plates the same final concentrations were usedand volumes were adapted. If 96-well plate results were available theseresults were incorporated in Table-2, otherwise the results from the384-well plate assay were shown.

TABEL 2 in vitro filter in vitro SPA in vitro SPA assay assay assayCompound PARP-1 PARP-1 TANK-2 No pIC50 pIC50 pIC50 1 8.11 <5 2 6.0126.876 <5 3 6.272 5.753 4 5.438 6.144 <5 5 5.579 6.195 <5 6 5.563 6.412<5 7 5.464 6.228 6.127 8 5.676 6.272 <5 11 <5 12 <5 13 <5 16 <5 17 <5 185.345 6.072 <5 19 6.204 7.498 <5 20 5.276 6.171 <5 21 6.284 7.593 <5 226.331 7.334 5.073 23 5.595 6.163 <5 24 5.305 6.105 <5 25 5.635 6.721 <526 5.789 6.372 <5 27 6.373 7.353 5.099 28 5.55 5.827 <5 29 5.333 6.105<5 30 7.491 6.1 31 7.405 <5 32 7.345 33 7.458 6.028 34 7.664 35 7.971 367.965 <5 37 7.816 <5 38 6.373 6.27 39 8.003 <5 40 5.649 6.492 <5 416.263 7.352 <5 42 5.048 6.075 <5 43 7.908 <5 44 7.533 <5 45 5.516 6.647<5 46 5.729 6.637 <5

The compounds can be further evaluated in a cellular chemo- and/orradiosensitization assay, an assay measuring inhibition of endogenousPARP-1 activity in cancer cell lines and eventually in an in vivoradiosensitization test.

1. A compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein X is >N—]or >CH—;—N═Y— is —N—C(O)— or —N═CR⁴—, wherein R⁴is hydroxy; L is a direct bondor a bivalent radical selected from —C(O)—, —C(O)—NH—, —NH—,—C(O)—C₁₋₆alkanediyl-, —C(O)—O—C₁₋₆alkanediyl- or —C₁₋₆alkanediyl-; R¹is hydrogen, halo, C₁₋₆alkyloxy or C₁₋₆alkyl; R² is hydrogen, hydroxy,C₁₋₆alkyloxy or aminocarbonyl; when X is substituted with R² thanR²taken together with -L-Z can form a bivalent radical of formula—C(O)—NH—CH₂—NR¹⁰—  (a-1) wherein R¹⁰ is phenyl; R³ is hydrogen, orC₁₋₆alkyloxy; Z is amino, cyano or a radical selected from

wherein each R⁵, R⁶, R⁷ and R⁸ is independently selected from hydrogen,halo, amino, C₁₋₆alkyl or C₁₋₆alkyloxy; or R⁷ and R⁸ taken together mayform a bivalent radical of formula—CH₂—CR⁹ ₂—O—  (c-1),—(CH₂)₃—O—  (c-2),—O—(CH₂)₂—O—  (c-3) or—CH═CH—CH═CH—  (c-4) wherein each R⁹ is independently selected fromhydrogen or C₁₋₆alkyl; with the proviso that when X is >N—, then Z isother than the radical (b-2) and when X is >CH— and L is —C(O)—NH— or—C(O)—O—C₁₋₆alkanediyl- and Z is the radical (b-2) and R⁷ and R⁸ takentogether form a bivalent radical of formula (c-1), (c-2) or (c-3) thenR⁵ is other than chloro.
 2. A compound as claimed in claim 1 wherein Lis a direct bond or a bivalent radical selected from —C(O)—, −C(O)—NH—,or —C(O)—O—C₁₋₆alkanediyl-; R² is hydrogen, hydroxy, or C₁₋₆alkyloxy; Zis a radical selected from (b-2), (b-3), (b-4), (b-5), (b-6), (b-7),(b-8) or (b-9); each R⁵, R⁶, R⁷ and R⁸ is independently selected fromhydrogen, halo, amino, C₁₋₆alkyl or C₁₋₆alkyloxy; or R⁷ and R⁸ takentogether may form a bivalent radical of formula (c-1), (c-2), (c-3) or(c-4).
 3. A compound according to claim 1 wherein L is a direct bond; R¹is hydrogen, halo or C₁₋₆alkyl; R² is hydrogen; R³is hydrogen; Z is aradical selected from (b-5) or (b-7); and each R⁵ is independentlyselected from hydrogen or halo.
 4. A compound selected from the groupconsisting of:


5. (canceled)
 6. A pharmaceutical composition comprisingpharmaceutically acceptable carriers and as an active ingredient atherapeutically effective amount of a compound as claimed in claim
 1. 7.A process of preparing a pharmaceutical composition as claimed in claim6 wherein the pharmaceutically acceptable carriers and the compound asclaimed in claim 1 are intimately mixed.
 8. A method of treating a PARPmediated disorder in a subject, said method comprising administering tothe subject a therapeutically effective amount of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein the dotted linesrepresent optional bonds; X is >N— or >CH—; —N═Y— is —N—C(O)— or—N═CR⁴—, wherein R⁴is hydroxy; L is a direct bond or a bivalent radicalselected from —C(O)—, —C(O)—NH—, —NH—, —C(O)—C₁₋₆alkanediyl-,—C(O)—O—C₁₋₆alkanediyl- or —C₁₋₆alkanediyl-; R¹ is hydrogen, halo,C₁₋₆alkyloxy or C₁₋₆alkyl; R² is hydrogen, hydroxy, C₁₋₆alkyloxy oraminocarbonyl; when X is substituted with R² than R²taken together with-L-Z can form a bivalent radical of formula—C(O)—NH—CH₂—NR¹⁰—  (a-1) wherein R¹⁰ is phenyl; R³ is hydrogen, orC₁₋₆alkyloxy; Z is amino, cyano or a radical selected from

wherein each R⁵, R⁶, R⁷ and R⁸ is independently selected from hydrogen,halo, amino, C₁₋₆allkyl or C₁₋₆alkyloxy; or R⁷ and R⁸ taken together mayform a bivalent radical of formula—CH₂—CR⁹ ₂—O—  (c-1),—(CH₂)₃—O—  (c-2),—O—(CH₂)₂—O—  (c-3) or—CH═CH—CH═CH—  (c-4) wherein each R⁹ is independently selected fromhydrogen or C₁₋₆alkyl.
 9. The method of claim 8 wherein the PARPmediated disorder is a PARP-1 mediated disorder.
 10. The method of claim8, wherein the treatment involves chemosensitization.
 11. The method ofclaim 8, wherein the treatment involves radiosensitization.
 12. Acombination of a compound with a chemotherapeutic agent wherein saidcompound is a compound of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein the dotted linesrepresent optional bonds; X is >N— or >CH—; —N═Y— is —N—C(O)— or—N═CR⁴—, wherein R⁴is hydroxy; L is a direct bond or a bivalent radicalselected from —C(O)—, —C(O)—NH—, —NH—, —C(O)—C₁₋₆alkanediyl-,—C(O)—O—C₁₋₆alkanediyl- or —C₁₋₆alkanediyl-; R¹ is hydrogen, halo,C₁₋₆alkyloxy or C₁₋₆alkyl; R² is hydrogen, hydroxy, C₁₋₆alkyloxy oraminocarbonyl; when X is substituted with R² than R²taken together with-L-Z can form a bivalent radical of formula—C(O)—NH—CH₂—NR¹⁰—  (a-1) wherein R¹⁰ is phenyl; R³ is hydrogen, orC₁₋₆alkyloxy; Z is amino, cyano or a radical selected from

wherein each R⁵, R⁶, R⁷ and R⁸ is independently selected from hydrogen,halo, amino, C₁₋₆allkyl or C₁₋₆alkyloxy; or R⁷ and R⁸ taken together mayform a bivalent radical of formula—CH₂—CR⁹ ₂—O—  (c-1),—(CH₂)₃—O—  (c-2),—O—(CH₂)₂—O—  (c-3) or—CH═CH—CH═CH—  (c-4) wherein each R⁹ is independently selected fromhydrogen or C₁₋₆alkyl.
 13. A process for preparing a compound as claimedin claim 1, said process comprising: reacting an intermediate of formula(II) with an intermediate of formula (III), wherein W is an appropriateleaving group, with the formation of a compound of formula (1-a),wherein L¹ is —C₁₋₆alkanediyl-NH— and both dotted lines can be a bond,in a reaction-inert solvent and with the addition of an appropriatebase,


14. A pharmaceutical composition comprising pharmaceutically acceptablecarriers and as an active ingredient a therapeutically effective amountof a compound as claimed in claim
 2. 15. A pharmaceutical compositioncomprising pharmaceutically acceptable carriers and as an activeingredient a therapeutically effective amount of a compound as claimedin claim
 3. 16. A pharmaceutical composition comprising pharmaceuticallyacceptable carriers and as an active ingredient a therapeuticallyeffective amount of a compound as claimed in claim
 4. 17. A method oftreating a PARP mediated disorder in a subject, said method comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim
 2. 18. A method of treating a PARPmediated disorder in a subject, said method comprising administering tothe subject a therapeutically effective amount of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of claim
 3. 19. A method of treating a PARP mediated disorderin a subject, said method comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound of claim4.
 20. The method of claim 18, wherein the PARP mediated disorder is aPARP-1 mediated disorder.
 21. The method of claim 19, wherein the PARPmediated disorder is a PARP-1 mediated disorder.